Serological tests for circulating tumor markers (biomarkers), such as PSA and AFP, are simple and cost-effective diagnostic and prognostic tests. However, only a handful of useful serum tumor marker tests are available clinically. Recent clinical proteomics indicates that specific biomarkers or protein signatures are present in the serum even during early cancer development. However, these biomarkers are present in the serum in low levels and likely beyond the detection limit of conventional ELISA. The principle investigators have invented a new antigen detection and quantification method termed Fluorescent Amplification Catalyzed by T7 RNA polymerase Technology (FACTT). FACTT uses similar principles as ELISA but coupled with the linear amplification ability of T7 RNA polymerase. It is a high throughput isothermal quantitative antigen detection assay and the preliminary data showed that FACTT increased the detection limit of ELISA by three or more orders of magnitude. FACTT assays are developed using 96 well or 384 well plates and can be easily used in clinical chemistry laboratory. As a proof of principle, this application will focus on developing FACTT assays to detect circulating biomarkers associated with malignant melanomas, because melanomas have cell lineage specific markers that can be targeted, but useful serological tests are lacking. The Pis plan to: 1) Set up and optimize FACTT assays to known melanoma markers (tyrosinase, TRP-1 and Melan-A) using pure proteins or cell lysates as template; 2) Optimize the FACTT assays in serum samples and test robustness and reproducibility of these assays; and then quantify tumor markers in sera collected from patients with measurable metastatic melanoma. 3) Validate specificity of the FACTT assays and establish normal ranges of the analytes in large cohorts of control population. The detection of circulating biomarkers in the early stage of tumor progression using FACTT assays would allow clinicians to identify high risk patients, to stratify patients for specific treatment, and to monitor treatment efficacy and disease recurrence. Early detection of metastatic melanoma might spur adjuvant therapies that could help eradicate metastases before they become clinical evident. With the emerging treatment options for melanoma such as Braf inhibitors and immunotherapies, there is a strong need for ultrasensitive assays to detect early melanoma metastasis. [unreadable] [unreadable] ASSESSMENT: [unreadable] [unreadable] [unreadable]